The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, of which the Toll-like Receptors (TLRs) play a prominent role. Unlike the antigen receptors of acquired immunity, the TLRs are encoded by a limited number of germline genes, ten in humans; however, in spite of their small numbers, the TLRs recognize a remarkably wide variety of PAMPs including glycolipids, proteins, and nucleic acids. We have been investigating several aspects of TLR structure and function: 1. The molecular basis for the recognition of a wide array of PAMPs by TLRs is a main interest of my laboratory. In collaboration with Dr. David Davies (LMB, NIDDK), we have recently succeeded in expressing, crystallizing, and determining the molecular structure of the TLR3 extracellular domain (ECD). The structure is quite elegant; it consists of a solenoid of 23 turns, bent into a horseshoe shape, with a large beta-sheet on the concave surface. The molecules is heavily glycosylated, except that, remarkably, one lateral face of the horseshoe is totally devoid of glycan. Although we have not yet obtained a high resolution structure of a TLR3-ligand (dsRNA) complex, we have located the dsRNA binding site by mutational analysis. The ligand binds on the glycan-free lateral face of the TLR3 molecule, near the C-terminal end, and we have proposed a model that would explain how dsRNA interacts and activates TLR3. We are also studying the binding of various oligomers of dsRNA to TLR3-ECD protein in solution to determine the affinity, specificity, and kinetics of ligand binding. Looking past TLR3, we plan to express and examine ECDs from other TLR paralogs, to see how they differ in structure and ligand binding function from TLR3. 2. Nucleic acid PAMPs such as dsRNA, ssRNA, and CpG DNA, ligands for TLRs 3, 7, 8, and 9, are normally sequestered within microorganisms and become available to interact with TLRs only after the pathogen is endocytosed and lysed intracellularly. By contrast, TLRs 1, 2, 4, 5, 6, and 10 interact with PAMPs that are normally present in the medium, and these TLRs are, as expected, located on the cell surface. Therefore, correct cellular localization is essential for TLR function. We are currently studying the localization of TLR9, and motifs within the TLR9 molecule that mediate this localization. We have found that TLR9 is located, prior to stimulation, in the ER, and that it interacts with CpG DNA in early endosomes. Both the cytoplasmic and extracellular domains contain internalization signals, and we have also located, within the cytoplasmic domain of TLR9 two regions that control intracellular localization. We are especially interested in neutralizing the internalization signal on the extracellular domain, so that we will be able to generate large amounts of secreted TLR9-ECD for structural studies. Future studies will also be directed to examining the intracellular location of TLR3. 3. TLRs play a pivotal role in acquired immunity by triggering the maturation of DC to competent APC, capable of priming naive T cells. Based on our observation that DC also express histamine receptors, we hypothesized that histamine would have an effect on the maturation process. In testing this hypothesis we found that histamine profoundly alters the cytokines released by DC during TLR induced maturation, and as a result, histamine exposure causes DC to polarize naive T cells toward a Th2 phenotype. Mast cells are the major source of histamine, and they are often located in close proximity to DC.